The goals of this application are to develop the concepts of degenerate recognition in nucleic acid enzymology as a general strategy for understanding or manipulating biological systems. The hypothesis under consideration is that the electronic tuning of a set of isosteric and conformationally ambiguous nucleobases can result in useful probes of selectivity in protein-nucleic acid interactions. In the course of testing this hypothesis, we will explore opportunities to refine chemical probes of DNA polymerases and expand the tools for basic biochemical studies as well as enhance biotechnology. The opportunities for applying the principle of degenerate recognition will be also explored in the context of RNA dependent viruses that pose serious threats to human health. A team of investigators are assembled that will enable an effective combination of chemical, biochemical, and biophysical methods to address the molecular, structural and dynamic properties of the RNA and DNA polymerases. By focusing on the application of novel biochemical tools to investigate important biological systems, we aim to advance both our understanding of molecular biology, and the characterization of potential therapeutic targets. The goals of this proposal are: 1. To further develop azole carboxamide nucleobases as mechanistic probes of Pol I DNA polymerases. 2. To expand the technology base for azole carboxamide nucleotide triphosphate incorporation and strand extension using DNA polymerases. 3. To implement azole nucleobases as random mutagens to study the molecular evolution of 5- aminoimidazole ribonucleotide carboxylase (AIRC), an enzyme in de novo purine metabolism. 4. To develop chemoenzymatic methodologies for creating RNA templates containing azole carboxamide nucleobases and to analyze these templates in their recognition by RNA dependent polymerases.